Download Characterization of Lamprey Fibrinopeptides

Biochem. J. (1965) 94, 742
742
Characterization of Lamprey Fibrinopeptides
BY R. F. DOOLITTLE*
Coagulation Re8earch Laboratory, Chemi8try Department II, Karolin8ka In8titutet,
Stockholm, Sweden
(Received 7 September 1964)
1. Lamprey fibrinopeptide B is a relatively large peptide made up of about 40
amino acid residues. The peptide is highly electronegative, containing a large
number of aspartic acid residues and a tyrosine 0-sulphate residue. 2. The amino
acid sequence of the first 18 residues from the N-terminal end of fibrinopeptide B
has been established. The C-terminal ends with the sequence Val-Arg. Fibrinopeptide B is released by both lamprey and bovine thrombins. 3. Lamprey fibrinopeptide A is a short peptide containing only eight residues. The proposed amino
acid sequence is:
Asp-Asp-Ser-Ile/Leu-Asp-Ser-Leu/Ile-Arg
This peptide is released by lamprey thrombin but not by bovine thrombin.
Evidence has been submitted showing that at
least two fibrinopeptides are released from lamprey
fibrinogen by lamprey thrombin (Doolittle, 1965).
Only one of these is released by bovine thrombin
during its transformation of the lamprey fibrinogen
into fibrin. Characterization of these fibrinopeptides represents a first step in attempting to find
out why and how the specificities of the two
thrombins differ. In the present paper results are
presented to show that both fibrinopeptides have
arginine residues at the C-terminal end. Since
only N-terminal glycine groups appear during the
transformation of lamprey fibrinogen into fibrin, it
is evident that only arginyl-glycine bonds are
split during the release of both peptides. Fibrinopeptide A, which is not released by the bovine
thrombin, has aspartic acid as its N-terminal
residue. Fibrinopeptide B has glutamic acid as
its N-terminal residue and is split off by both
thrombins. Fibrinopeptide B is shown to be
definitely homologous to mammalian fibrinopeptides B, since it contains the unusual amino
acid derivative tyrosine 0-sulphate, an amino acid
found in a variety of mammalian fibrinopeptides B.
Further, the amino acid sequence in the area of the
sulphated tyrosine is identical with that found in
mammalian fibrinopeptides B.
MATERIALS AND METHODS
Lamprey thrombin and fibrinogen. Full details on the
purification of lamprey thrombin and fibrinogen, as well as
* Present address: Department of Biology, University
of California, San Diego, Calif., U.S.A.
for the clotting of the fibrinogen by lamprey or bovine
thrombin, have been presented by Doolittle (1965).
Column chromatography. Dowex 50 (X2; 200-400 mesh)
ion-exchange resin was pretreated according to the method
of Hirs, Moore & Stein (1956). Columns were equilibrated
with 0.1 m-ammonium formate buffer, pH 3 0. Peptides
were eluted by stepwise changes of buffers in the direction of
higher pH. In the region pH 40-7 0, 0.1 M-ammonium
acetate buffers were used. Dowex 1 (X2) was converted
first into the OH- form with N-NH3 and then into the
acetate form with acetic acid. It was finally equilibrated
with 0-1 M-ammonium acetate buffer, pH 5*5. Stepwise
elution was accomplished by changes to appropriate
volatile buffers at lower pH. Fractions were freeze-dried
and samples examined for peptides in the paper-electrophoresis system. Sephadex G-25 (Pharmacia, Uppsala,
Sweden) columns were poured after allowing the gel
particles to swell in 1% NaCl solution followed by extensive
washing with distilled water. Fine particles were removed
by decantation. The Sephadex columns were equilibrated
and developed with 0-05 m-pyridine. Column development
was monitored in all cases by subjecting samples from each
fraction to alkaline hydrolysis followed by ninhydrin
analysis according to the method of Moore & Stein (1954a,b).
Amino acid determination8. Peptides were hydrolysed in
twice-distilled 5 7 N-HCl at 1100 for 20 hr. Sample tubes
were thoroughly flushed with nitrogen gas before sealing.
The HCI was removed under reduced pressure in a desiccator
containing NaOH pellets. If the samples were to be subjected to quantitative measurement by column chromatography, a volume of water equal to the original volume of
the hydrolysis solution was added to the sample and the
drying operation repeated three times. If the samples were
to be subjected to qualitative analysis by combination
paper electrophoresis-paper chromatography, the rinsing
procedure was not necessary. Instead, the residues were
dissolved in 25 pl. of water and applied to a spot in the
centre of 4 cm.-wide paper-electrophoresis strips (Schleicher
Vol. 94
LAMPREY FIBRINOPEPTIDES
and Schull no. 2043). Electrophoresis was conducted in
0.1 M-pyridine-acetic acid buffer, pH 4-1, at 300 v for 4 hr.
at room temperature. Papers were dried in an oven at 800
and then stitched to Whatman 3MM paper for ascending
chromatography in butan-l-ol-acetic acid-water (4:1:5,
by vol.) for 17 hr. After being thoroughly dried, papers
were stained with McMenamy's differential-colour ninhydrin reagent (McMenamy, Lund & Oncley, 1957).
Alkaline hydrolysis was conducted in 0-2 M-Ba(OH)2 at
115-120° for 20 hr. The Ba(OH)2 solution was made up
with hot water and filtered with a stemless funnel just
before use. Tubes were sealed immediately. The hydrolysate (usually 50 ,ul.) was applied directly to the paperelectrophoresis strips for analysis by combination paper
electrophoresis-paper chromatography. The applications
were made to the dry papers, and the buffer was allowed to
approach from each extremity. A smooth gel tends to form
at the spot of application of the Ba(OH)2 solution, but this
does not influence the subsequent migration of amino acids.
In those experiments in which it was desirable to identify
tyrosine 0-sulphate, the pH was lowered to 3 9 to effect
better separation of the tyrosine 0-sulphate from aspartic
acid. Reference tyrosine 0-sulphate was a gift from
Dr B. Blombiick. Quantitative analyses ofacid hydrolysates
of fibrinopeptides A and B were performed on a microchromatographic apparatus fashioned and operated by
A. Baldesten. The single-column (Amberlite IR-120)
development is based on the method of Spackman, Stein
& Moore (1958), but it is possible to measure amino
acids with reasonable accuracy at the level of 0-02
,umole. The column (0 3 cm. x 175 cm.; resin particle size,
20-30 ,) was operationally similar to that described by
Hamilton (1963). Samples for analysis were dissolved in
a pH 2 2 buffer, and the initial elution was carried out
at pH 3 0. The appearance of the glutamic acid peak on
the recorder served as a signal to shift the elution over to
a gradient system beginning at pH 3 46 and increasing to
pH 6 0. The author is indebted to A. Baldesten and N. R.
Kale for designing the apparatus used.
Enzymic degradation of peptide8. Twice-recrystallized
trypsin and chymotrypsin were dissolved in 0-1 MNH4HCO3 buffer, pH 8 0, usually at a concentration of
0.1 mg. of enzyme/ml. of buffer. The enzyme:substrate
ratio was about 1: 50 on a weight basis. Incubations were
carried out at 370 for 5 hr. Samples, and the appropriate
enzyme blanks and controls, were applied directly to
4 cm.-wide paper-electrophoresis strips (dry loading),
and subjected to electrophoresis in 01 m-pyridine-acetic
acid buffer, pH 4-1, at 300 v for 4 hr. Guide strips about
2 mm. wide were cut from each paper and stained with the
McMenamy ninhydrin stain. Areas corresponding to the
ninhydrin-positive bands were eluted with water, the
material was freeze-dried and hydrolysed in acid, and the
qualitative amino acid compositions were determined.
Alternatively, in some experiments the digests were streaked
on chromatography paper (Whatman no. 1) and subjected
to descending chromatography in butan-l-ol-acetic acidwater (4:1:5, by vol.). Carboxypeptidase A (Sigma
Chemical Co., St. Louis, Mo., U.S.A.) and carboxypeptidase
B (Worthington Biochemical Corp., Freehold, N.J.,
U.S.A.) were also used in 041 M-NH4HCO3 buffer, pH 8-0.
Neither of the preparations was treated with di-isopropyl
phosphorofluoridate, and the carboxypeptidase B preparation was found to contain chymotryptic and carboxy-
743
peptidase-A activities. Incubations were carried out at
room temperature, and samples were removed periodically
and spotted or streaked directly on paper strips for analysis
by combination paper electrophoresis-paper chromatography.
Subtilisin was a generous gift from S. Paleus. The enzyme
was dissolved in 0.1 m-NH4HCOs buffer, pH 8-0, at a
concentration of 0-2 mg./ml. of buffer and the digestions
were carried out for 18 hr. at room temperature. The
entire incubation mixture was 'dry-loaded' on to paperelectrophoresis strips and electrophoresis conducted in
0.1 m-pyridine-acetic acid buffer, pH 5X6, at 220 v for 5 hr.
Guide strips were stained and the areas corresponding to
the ninhydrin-positive bands eluted as described above.
Partial acid hydrolysis. The enzymic studies were
supplemented by partial-hydrolysis studies with 1%
acetic acid, pH 2-8, in sealed tubes at 1100 for 16 hr. Only
aspartic acid links were appreciably split with this method
(cf. Blackburn & Lee, 1954). It was found convenient to
'dry-load' the partial hydrolysates directly on to electrophoresis papers. They were analysed for free amino acids
and peptides in the combination system and also were run
in the electrophoresis system alone for the elution of
components.
Edman degradations. The Edman (1950) reaction was
carried out in its three-cycle form (Edman, 1960) in an
experimental manner designed by P. Edman (unpublished
modification). Samples for degradation ranging from
3 to 15 mg. were dissolved in 1 0 ml. of a 0 4 m-dimethylallylamine buffer, pH 9-6 (15-0 ml. of pyridine, 10.0 ml. of
water and 1-18 ml. of dimethylallylamine). Then 50 pJ. of
distilled phenyl isothiocyanate was added and the pH of the
reaction mixture adjusted to 9 0 with dilute aqueous
trifluoroacetic acid. The dipping electrode of the Radiometer pH-meter was rinsed with a few drops of dimethylallylamine buffer, pH 9 0, and the rinsings were added to
the reaction mixture. The tubes were flushed for a few
seconds with nitrogen, stoppered with glass and incubated
at 400 for 1 hr. The mixture was then washed thoroughly
with 2-4 ml. of benzene five times, the organic phases
being discarded. The last traces of benzene were removed
with a stream of nitrogen. About 0 5 ml. of water was
added to the aqueous phase and the system freeze-dried
from a desiccator containing NaOH and P205 for at least
2 hr. The resulting residues were carefully washed three
times with 0.5 ml. of ethyl acetate each time. Final traces
of ethyl acetate were removed by gentle aspiration and then
by placing the tubes in a desiccator under reduced pressure.
The phenylthiocarbamoyl derivatives of the peptides
were then dispersed in a minimal volume (25-100 ,ul.) of
anhydrous trifluoroacetic acid and the tubes gently flushed
with nitrogen. The glass-stoppered tubes were placed at
400 for 15 min. for the cyclization and cleavage mechanism
to be completed. The resulting thiazolinones were extracted
with 2-4 ml. of ethylene chloride, the extractions being
performed twice on each sample. The ethylene chloride
was evaporated in a stream of nitrogen and the thiazolinones
were converted into the corresponding phenylthiohydantoins according to the method of Ilse & Edman (1963). The
phenylthiohydantoins were dissolved in ethylene chloride,
samples were removed and diluted with ethanol, and
spectra were recorded on a Beckman DK-2a ratio recording
spectrophotometer. Yields were calculated on the basis of
the proper extinction coefficient for the amino acid sub-
R. F. DOOLITTLE
744
sequently identified (Sjoquist, 1957). Appropriate amounts
of the PTH-amino acids* were run in the D, E, F solvent
system of Edman & Sj6quist (1956). Valine was distinguished from phenylalanine in solvent I of the system of
Sjdquist (1960). Chromatograms were examined for ultraviolet-absorbing spots and were then sprayed with the
iodine-azide reagent (Sjoquist, 1953). Degradative steps
on a given peptide were accomplished at the rate of one per
day. The peptide chains were stored in a desiccator at 40 at
all times except when being treated, washed or degraded.
Certain glutamic acid residues were verified by alkaline
hydrolysis of the PTH-amino acid to the free amino acid
with 02 M-Ba(OH)2 at 1200 in sealed tubes for 20 hr.
Identification was made by paper electrophoresis. Glutamine and asparagine residues were verified by removing
samples of the PTH-amino acids and subjecting them to
mild hydrolysis (1 x-HCl at 1000 for 1 hr.). The sample was
then rechromatographed and the phenylthiohydantoin of
either glutamic acid or aspartic acid identified, as the case
may have been. In all cases the reagents were washed or
redistilled or both, as specified (Edman & Sj8quist, 1956).
Miscellaneous methods. The Sakaguchi reagent was
prepared and administered as directed by Acher & Crocker
(1952). Ninhydrin-negative material was detected by the
chlorine gas method (Rydon & Smith, 1952). An aniline
phthalate spray was used to examine peptides for carbohydrate (Partridge, 1949), and an Ehrlich's test for indoles
was conducted with p-dimethylaminobenzaldehyde (Reddi
& Kodicek, 1953). The iodine-azide reagent was used as a
test for thiol compounds (Chargaff, Levine & Green, 1948).
RESULTS
Fibrinopeptide B
Purifiation. The isolation of lamprey fibrinopeptide B was unknowingly facilitated by clotting
lamprey fibrinogen with bovine thtombin. In the
best preparations of fibrinogen (from batch I
plasma; Doolittle, 1965), the freeze-dried clot
liquors needed only to be passed over Sephadex
G-25 to be pure enough for direct sequence studies.
The concentrated material gave only one band on
electrophoresis at pH 4-1, and no other components
were revealed when the paper strip was stitched tc
chromatography paper and subjected to ascending
chromatography. The band was ninhydrin-positive
and Sakaguchi-positive, and gave a strong reaction
with the chlorine gas method of detection. The
material was negative to aniline phthalate as a
test for carbohydrate, gave a negative Erlich's
test and was negative for thiol compounds as
determined by spraying with the iodine-azide
reagent. The peptide had an RF less than 0-02 in
butan-l-ol-acetic acid-water (4:1:5, by vol.).
Preparations of fibrinopeptide B were also
obtained by direct elution from paper strips after
electrophoresis of freeze-dried clot liquors. Larger
quantities were prepared by adsorption from the
* Abbreviation: PTH-amino acids,
phenylthiohydantoin
derivatives of amino acids.
1965
O Leu/lle
O Val
8 Tyr
Pro
C
OGIU
Glu
TAla
Thr
Gly
Asp
~Ser
Arg
)Lys
Fig. 1. Tracing of amino acids found on combination paper
electrophoresis-paper chromatography after total acid
hydrolysis of lamprey fibrinopeptide B. Material was
applied at the spot marked by a cross (+) and subjected to
electrophoresis at pH 4-1. The paper strips were dried and
then stitched to chromatography paper for ascending
chromatography in butan-l-ol-acetic acid-water (4:1:5,
by vol.) for 17 hr. After being thoroughly dried, papers
were stained with differential-colour ninhydrin reagent: Y,
yellow; Br, brown; all other spots various shades of blue.
Full details are given in the text.
concentrated clot liquors on to Dowex 1 (X2)
columns (0.9 cm. x 70 cm.) at pH 4*5 and elution
at pH 2-5. Fibrinopeptide B could not be adsorbed
on Dowex 50 (X2) as can many mammalian fibrinopeptides (cf. Blombaick & Vestermark, 1958),
presumably because of its unusually large size and
very high electronegativity.
Amino acid composition. The qualitative amino
acid composition of lamprey fibrinopeptide B, as
well as its electrophoretic mobility on paper strips,
was consistent with that reported for the lamprey
'major' fibrinopeptide by Doolittle, Oncley &
Surgenor (1962), except that at least two additional
amino acids (tyrosine and lysine) were detected
after acid hydrolysis (Fig. 1). Quantitative amino
acid analysis on the micro-chromatographic system
indicated that the peptide contained about 40
amino acid residues (Table 1), the previously
undetected amino acids occurring only once each.
The peptide contains no phenylalanine, histidine,
cysteine, tryptophan or methionine. One residue
of isoleucine was identified in the peptide from one
batch of lamprey plasma (batch I), but not in
another from a different source (batch II). Measurements of amide nitrogen were not made, but the
ammonia peak on chromatographic analysis suggested that some of the aspartic acid and glutamic
acid residues existed as asparagine and glutamine
respectively.
Alkaline hydrolysis revealed that the unusual
Vol. 94
LAMPREY FIBRINOPEPTIDES
Table 1. Amino acid compo8ition of lamprey
fibrinopeptide B
Amino acids are listed in the order in which they emerge
from column. Molar ratios were computed by taking the
value for aspartic acid to be 13-00. Threonine and serine
were corrected for losses during acid hydrolysis (5 and 10%
respectively). Preparation II (from batch I plasma) was
purified on Dowex 1 (X2). Preparation IV (from batch II
plasma) was purified by elution from paper-electrophoresis
strips. In preparation IV, the lysine peak was occluded by
the ammonia peak, but the presence of lysine in the peptide
was confirmed by paper electrophoresis-paper chromatography. The analyses of preparations II and IV differed
significantly with regard to the number of alanine, valine
and isoleucine residues.
Preparation IV
Preparation II
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Molar
ratio
13-00
2-90
2*51
2-79
2-15
2*04
4-05
2-98
1-13
3*64
0 93
0-64
0-87
Isoleucine
Leucine
Tyrosine
Lysine
Arginine
Total residues ...
Whole
numbers
13
3
2-3
3
2
2
4
3
1
3-4
1
1
1
39-41
Molar
ratio
13-00
2*87
1X76
2-92
1X61
2X10
6-38
1-92
0
3.54
0.90
0X79
Whole
numbers
13
3
2
3
2
2
6
2
0
3-4
1
(1)
1
39-40
Leu/Ile
Val
y
.Pro
Tyr 0-sulphate Glu
Br
Ala
Thr
D Gly®{
Asp
Lys
Fig. 2. Tracing of amino acids found after alkaline hydrolysis of lamprey fibrinopeptide B. The hydrolysate was
applied directly to a paper strip, and combination paper
electrophoresis-chromatography was conducted as indicated in Fig. 1 except that the pH was adjusted to 3 9 to
improve the separation of tyrosine 0-sulphate. Free
tyrosine was absent. A reference preparation of tyrosine
0-sulphate was run simultaneously on a companion paper.
(a)
745
(b)
rn
.
B
Origin-I
(d)
(c)
lT.
E
Asp
Ch-l
_
o0I
Ch-ll
I
v,iS-111
y
HA-I
S
S-Ill
S-Iv
Origin
I-S-V
Fig. 3. Paper-electrophoresis strips of various lamprey
fibrinopeptide B digestions. (a) Undigested fibrinopeptide
B; (b) after digestion with chymotrypsin (Ch); (c) splitting
of Ch-II with 1% acetic acid (HA); (d) subtilisin (S)
digestion of Ch-I. Cross-hatched areas, strong blue colour
with ninhydrin reagent; stippled area, weak blue colour;
Y, yellow. Electrophoresis was carried out at pH 4-1 for
(a)-(c), and at pH 5-6 for (d).
derivative tyrosine 0-sulphate is present in fibrinopeptide B (Fig. 2). When the peptide is hydrolysed
in this way, no free tyrosine is found. The unhydrolysed peptide does not exhibit an absorption peak
in the ultraviolet region 270-300 m,u. Except for
the shift of the tyrosine spot to the position of
tyrosine 0-sulphate, paper chromatograms of
alkaline hydrolysates were qualitatively the same
as those of acid hydrolysates.
Enzymic digestion. Lamprey fibrinopeptide B
was not split by trypsin under the conditions
described, in spite of the fact that the molecule
appears to contain one lysine residue that is neither
N- nor C-terminal. Only one electrophoretic band,
identical with that of undigested material, was
found after incubation with the enzyme. Descending chromatography also exhibited only the
undigested peptide. Under the same conditions,
chymotrypsin split fibrinopeptide B into two
fragments (Fig. 3). One of these, a small peptide
containing arginine, valine and aspartic acid, was
isolated both from electrophoretic strips and from
paper chromatograms. The other piece after the
degradation tended to move with a mobility only
slightly greater than that of the undigested peptide.
Its RF on paper chromatography was also similar to
that of the undigested material. The two chymotryptic fragments are referred to below as Ch-I
(the large piece, deduced to be N-terminal) and
Ch-II (the small piece, deduced to be C-terminal).
Qualitative analysis of Ch-I indicated that arginine
was no longer present. Other than that, it was
qualitatively similar to the whole fibrinopeptide
B in its amino acid composition.
Intact fibrinopeptide B was resistant to the action
of carboxypeptidase A. The major fragment after
chymotryptic digestion, however, yielded two
746
R. F. DOOLITTLE
1965
Edman degradation. Direct sequence studies
with the Edman phenyl isothiocyanate method on
whole fibrinopeptide B were performed on three
separate preparations, all from batch I plasma.
A preliminary run (preparation I) indicated that
the N-terminal tripeptide was Glu-Asp-Ile. The
degradation was then carried out on two other
preparations, one of which had been purified by
stepwise elution from a Dowex 1 (X2) column
(preparation II) and the other only by passing the
concentrated clot liquor over Sephadex G-25
(preparation III). Preparation III came from a
clot liquor that had no detectable extinction at
280 m,u after clotting. The sequences of the first
seven residues split off by stepwise treatment were
identical in both preparations and confirmed the
terminal tripeptide found with preparation I.
Since preparations II and III appeared to have
identical sequences, and since only one amino acid
was detected at each step in both cases, the two
preparations were pooled and the degradation
continued through a total of 22 cycles. A total of
20 mg. of fibrinopeptide B was used in the degradations. On the basis of the amino acid composition
the molecular weight of the peptide was taken to be
approx. 4500. With allowances for salt and
moisture, a total of 3-4 ,umoles was used in these
studies. The stepwise results and yields are given
in Table 2. Yields are expressed as percentages of
the ,umoles found in the preceding step. The
average yield, exclusive of the first and last steps,
was 88%. Occasionally a yield would be unusually
low if, for example, the extracted thiazolinone was
stored in the freezer overnight as a convenience,
and not converted into the phenylthiohydantoin
until the next day. Since this was not at all related
to the state ofthe peptide remaining to be degraded,
the yield of the next step would usually be greater
than 100%. Since the yields were based on the
extinction of the phenylthiohydantoins before
I,,
chromatography, the possibility existed that nonspecific absorption in the ultraviolet region was
contributing to the high yields. For this reason,
the absorption spectra of several steps (11, 15 and
19) are represented in Fig. 5 as evidence that these
were true phenylthiohydantoins. Spectra were
i3Tyr
examined for all steps and found to be consistent.
Ala
The sequence was stopped, not because the yield
Asp
fell below detectable levels, but because the chains
0
were out of phase enough to preclude positive
0 0
identification of the 'new' N-terminal residue.
The glutamic acid residues at positions 1 and 10
were subjected to alkaline hydrolysis and the free
glutamic acid residues demonstrated. After preFig. 4. Tracing of combination paper electrophoresis- liminary identification on chromatography, the
PTH-glutamine at position 8 and the PTHpaper chromatography of 1% acetic acid digest of lamprey
fibrinopeptide B chymotryptic fragment Ch-I. The asparagine at position 11 were both treated with
mild acid and converted into PTH-glutamic acid
conditions were as given in Fig. 1.
amino acids, (leucine and alanine) after 60 min.
digestion with carboxypeptidase A. The leucine
residue is thought to be the C-terminal residue of
the fragment on the basis of the preferential
specificity of chymotryptic splitting. Degradation
of whole fibrinopeptide B with carboxypeptidase B
resulted in the appearance of arginine, valine,
leucine and alanine, but not aspartic acid. It was
assumed that the carboxypeptidase B preparation
contained chymotryptic activity as well as
carboxypeptidase A. Arginine was deduced to be
the C-terminal amino acid of fibrinopeptide B,
however, since carboxypeptidase A alone did not
degrade the peptide.
The N-terminal piece derived from chymotryptic
studies (Ch-I) was subjected to degradation by
-ubtilisin. At least five major ninhydrin-positive
bands were distinguished after paper electrophoresis (Fig. 3). This chymotryptic fragment
(Ch-I) was also partially hydrolysed with 1%
acetic acid. Free tyrosine and alanine were detected
in the hydrolysate, suggesting that these amino
acids had aspartic acid neighbours on both sides.
Large quantities of free aspartic acid were also
detected. A positively charged peptide was also
detected in the mixture after this treatment, as well
as an electronegative fragment with a very high
R. in butan-l-ol-acetic acid-water (Fig. 4).
The C-terminal piece resulting from chymotryptic digestion (Ch-II) was also studied by acetic
acid digestion. After the splitting, the peptide gave
only one positively charged fragment and free
aspartic acid. The positively charged fragment
contained only valine and arginine. Since the whole
fibrinopeptide contains only one arginine residue,
the deduced C-terminal dipeptidyl sequence is
Val-Arg.
LAMPREY FIBRINOPEPTIDES
Vol. 94
747
Table 2. Yields of phenylthiohydantoin derivatives of amino acid8 at each step of the sequence
in the degradation of lamprey fibrinopeptide
The amounts of PTH-amino acid were calculated on the basis of spectrophotometric measurements on samples
of the converted derivatives before chromatography. The yields are expressed as percentages of the material
(in ,umoles) found in the preceding step. Full details are given in the text.
Step
PTH-amino a cid
(utmoles)
1
2
3
4
5
6
7
1-47
1-10
1-02
0-97
1-00
0-95
0-64
8
9
10
11
12
0-90
0-56
0-66
0-70
0-47
0-43
0-46
0-34
0-29
0-29
0-31
0-23
13
14
15
16
17
18
19
20
21
22
0-19
0-19
0-29
ITraces
Result
Glu
74
Asp
93
Ile
95
Ser
103
Leu
95
Val
67
Gly
(Pooled with 0-3 pmole from another preparation)
94
(Gly, Glu)
Glu(NH2)
62
Pro
118
Glu
106
(Glu, Asp)
Asp(NH2)
67
Asp
[Asp(NH2 d)rI
92
Tyr
(Asp)
107
Asp
(Tyr)
74
Thr
(Asp, dehyydrothreonine)
85
Gly
100
(Gly)
Asp
107
Asp
(Gly)
74
Asp, Ala
83
Ala, Asp
(Thr)
100
Asp, Ala, Asp(NH2)
153
Asp, Ala
Yield (%)
and PTH-aspartic acid respectively. The isoleucine
at position 3 was identified on the basis of repeated
runs in solvent D (Edman & Sjbquist, 1956). In all
cases the sample moved with the isoleucine rather
than with the closely related leucine. The valine at
A
position 6 was originally identified in solvent D and
subsequently verified in Sjoquist's (1960)
solvent I. Serine was verified in step 4 by the
nature of the characteristic spectrum of its derivative before chromatography.
was
220
240
260
280
300
Wavelength (m,u)
320
340
Fig. 5. Absorption spectra of PTH-amino acids derived
from lamprey fibrinopeptide B: A, step 11; B, step 15;
C, step 19. In step 11, the phenylthiohydantoin was
dissolved in 150 pI. of ethylene chloride. In steps 15 and 19,
the preparations were dissolved in ll.
100
of ethylene
chloride. In each case 5 p,. of the ethylene chloride solution
was diluted with 1-0 ml. of ethanol for reading on the
Beckman DK-2a ratio recording spectrophotometer.
Step 11 was found to be asparagine, step 15 threonine and
dehydrothreonine, and step 19 a mixture of aspartic acid
and alanine (cf. Table 2).
Fibrinopeptide A
Isolation. Lamprey fibrinopeptide A was isolated
from the clot liquors of fibrin A preparations as well
as from fibrin B treated with lamprey thrombin
(Doolittle, 1965). In some cases the peptide was
eluted from paper strips after electrophoresis of
concentrated clot liquors or filtrates. In others it
was separated from the bulk of the fibrinopeptide B
or contaminating substances by extraction of the
freeze-dried clot liquor with 50% (v/v) propan-2-ol
or 75% (v/v) acetone. The propan-2-ol or acetone
extracts were evaporated to dryness, and the
R. F. DOOLITTLE
748
11
.0
I
0 .8.
~o *4.6
0
4'
0
0
1965
|EdmanH
%.........../
. ..
.2-A
k
L
7
5I
3
Cp
Asp-Asp-Ser-Ile/Leu-Asp-Ser-Leu/Ile-Arg
L
1
2
I
100 200 300 400 500
Vol. of effluent (ml.)
Fig. 6. Column chromatography on a Dowex 50 (X2)
column (0.9cm. x 60 cm.) of a 50% propan-2-ol extract of
freeze-dried filtrate obtained after treating lamprey fibrin
B (obtained with bovine thrombin) with lamprey thrombin.
Only free amino acids were found in peak I. Peak II was
lamprey fibrinopeptide A. -, Ninhydrin colour read at
570 mp; .... , pHE.
4
3
---
HA-1
5
<
6
7
--
HA-IIH
8
Fig. 7. Summary of sequence information concerned with
lamprey fibrinopeptide A. Edman, phenyl isothiocyanate
degradation; HA-I and HA-II, fragments obtained with
1% acetic acid partial hydrolysis; Cp, treatment of fibrinopeptide with carboxypeptidase B.
the three cycles, a peptide bearing a slight positive
charge at pH 4-1 was isolated from the degradation
material was redissolved in water and subjected to residue. The fragment contained aspartic acid,
chromatography on a Dowex 50 (X2) column serine, leucine or isoleucine and arginine. The
(0-9 cm. x 50 cm.). On stepwise elution, the peptide yield of PTH-aspartic acid released after the first
emerged from the column at pH 4-5 (Fig. 6). degradation of the fibrinopeptide A was 50% when
Preliminary separation of the peptide from the calculated on the basis of the molecular weight of
much larger fibrinopeptide B could also be accom- the octapeptide indicated by the quantitative
plished on Sephadex G-25, the small fibrinopeptide amino acid composition. This value, uncorrected
A being retained to a greater degree by the gel for salt and moisture, compared favourably with the
first-step yields obtained with ten mammalian
column (2-5 cm. x 40 cm.).
Amino acid coMpo8ition. Total acid hydrolysis fibrinopeptides isolated under similar conditions
revealed that the peptide contains only five different by BlombaLck & Doolittle (1963).
The deduced amino acid sequence of lamprey
amino acids. Its empirical amino acid composition
fibrinopeptide A is presented in Fig. 7. The relative
as determined on the micro-chromatographic
positioning of the leucine and isoleucine residues is
apparatus is ASp3, Ser2, Ilel, Leul, Argl.
Enzymic dige8tion8. Under the conditions des- as yet unknown, although the fact that the peptide
cribed above, the peptide was resistant to the action is resistant to the action of chymotrypsin suggests
of trypsin, chymotrypsin and carboxypeptidase A. that the leucine is penultimate to the C-terminal
Degradation with carboxypeptidase B for 30 min. end.
released free arginine and leucine or isoleucine (the
DISCUSSION
two amino acids could not be accurately distinThe characterization of the two lamprey fibrinoguished by combination paper electrophoresispeptides has been handicapped to a degree by the
paper chromatography). The mixture contained
only one other ninhydrin-positive substance, an limited amounts of material available for analysis.
electronegative peptide containing aspartic acid, Since the animals can only be caught during one
serine and leucine or isoleucine. The peptide month of a year during their spawning run, some
fragment was not Sakaguchi-positive, nor was equivocal judgements have to be made about how
to deploy a year's supply of material. In the
arginine found after acid hydrolysis.
Partial hydroly8i8 with acetic acid. Partial present report the preliminary characterization is
hydrolysis with 1% acetic acid resulted in free unusual in that considerably more material has
aspartic acid, one neutral peptide, and one positively been devoted to determining part of the sequence
charged peptide (pH 4-1). The neutral peptide of lamprey fibrinopeptide B by the direct Edman
contained serine and leucine or isoleucine; the method than has been allotted for precise quantitapositive fragment contained arginine, serine and tive amino acid analysis of the entire molecule.
These judgements were made concordant with the
leucine or isoleucine.
Edman degradation. Stepwise degradation of facilities available. Thus there is still some
1-9 mg. of fibrinopeptide A by the Edman phenyl doubt about the exact amino acid composition of
isothiocyanate method yielded a terminal tri- fibrinopeptide B (Table 1), since in two different
peptide sequence of Asp-Asp-Ser. At the end of analyses three of the 13 amino acids found after
Vol. 94
LAMPREY FIBRINOPEPTIDES
acid hydrolysis differed significantly on a molarratio basis. It is possible that the two different
preparations did differ slightly since they were
isolated from two different populations of animals.
On the other hand, the differences are not so great
that experimental variation in the purification and
micro-chromatographic analysis can be ruled out.
On the other hand, the N-terminal sequence of
fibrinopeptide B as established by the Edman
method is clear. The first 18 residues are unambiguous. In the last four degradative cycles, the
trailing from preceding steps made the assignment
of the new residues unclear. The emphasis on the
direct sequence method stemmed partly from
interest in the tyrosine 0-sulphate residue isolated
after alkaline hydrolysis. The residue had previously been found in six of seven mammalian
fibrinopeptides B (Bettelheim, 1954; Blomback,
Bostrom & Vestermark, 1960; Blomback & Doolittle, 1963), and there was hope of establishing a
definite homologous relationship between the
fibrinopeptide B of the primitive fish and those of
higher animals. If the sulphated tyrosine occurred
near the end of the chain, as it does in the known
mammalian fibrinopeptides B, then stepwise
degradation of the end of the peptide might provide
comparative data that would be conceivably more
useful than precise amino acid composition determinations. In fact, the single tyrosine residue
occurred in the thirteenth position from the
N-terminal end. It was preceded and followed by
aspartic acid residues, just as it is in all six of the
mammalian fibrinopeptides B that contain this
residue. This finding was consistent with the
results of acetic acid splitting, which released free
tyrosine. It seems probable that the tyrosine is
sulphated after its incorporation into the fibrinogen
chain, and that the sulphation, whether spontaneous or enzymic, demands the Asp-Tyr-Asp sequence.
This tripeptide sequence was the only one found in
the N-terminal region of the molecule that bore
any resemblance to those of mammalian fibrinopeptides, which are themselves highly variable.
It is unlikely that the tyrosine 0-sulphate plays
any role in thrombin attachment, since in the
lamprey fibrinopeptide it is considerably more
distant (in terms of number of residues) from the
vulnerable arginyl-glycine junction than is the
case in the mammalian fibrinopeptides. On the
other hand, the persistence of the sequence through
400 000 000 years of evolution suggests some
physiological role for the tyrosine 0-sulphate. It
may have a role either in fibrinogen dispersal or in
other phases of the haemostatic process after its
release from the parent fibrinogen molecule.
The fact that lamprey fibrinopeptide B is resistant to attack by trypsin may hinder the completion
of the amnino acid sequence determination. Splitting
749
with subtilisin, however, seems to offer a promising
method of attack.
WVhereas lamprey fibrinopeptide B is the largest
fibrinopeptide yet discovered, being roughly twice
as long as bovine fibrinopeptide B, lamprey
fibrinopeptide A is the smallest yet uncovered.
With the exception of the C-terminal arginine
residue, the peptide bears no resemblance to
mammalian fibrinopeptides A. The known mammalian fibrinopeptides A do have a great deal of
correspondence in parts of their amino acid
sequences, and the radical departure of the lamprey
fibrinopeptide may account for its not being
released by bovine thrombin. The extreme variability of the fibrinopeptides B that are released by
bovine thrombin argues against such reasoning,
however (cf. Doolittle & Blomback, 1964). The
most significant difference in sequence apparent
from comparisons of the lamprey fibrinopeptides
with their proposed mammalian homologues is
concerned with the amino acids at the second
position from the C-terminal end. In the mammalian fibrinopeptides A and B, only valine or
alanine is found at that position. In the lamprey
fibrinopeptide B, that position is occupied by
valine, and the fibrinopeptide is released by
bovine thrombin. In fibrinopeptide A, however,
that position is occupied by a leucine or isoleucine
residue. Since this position is adjacent to the link
that is attacked by thrombin, it is possible that the
larger leucine (or isoleucine) residue can block the
approach of the bovine thrombin. In terms of the
classical 'lock-and-key' hypothesis, it is not
difficult to visualize a valine fitting into a leucine
'hole', but the inverse may not be possible.
I express my gratitude to Professor E. Jorpes and Dr B.
Blombaick, in whose Laboratories these experiments were
conducted. I am especially grateful to my sponsor, Dr B.
Blomback, for his instruction in the use of the Edman
method, and to A. Baldesten for conducting the quantitative
amino acid analyses. The technical assistance of Mr P.
Nilsson and Miss Gunilla Croneborg is gratefully acknowledged. This investigation was supported in part by U.S.
Public Health Service Fellowship no. HE-14057, and by
Grant no. HE 7379-01, U.S. National Institutes of Health,
to Dr B. Blomback.
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